A Hb from the chloroplast of Chlamydomonas eugametos has been cloned andexpressed in E. coli. This Hb resembles several invetebrate in having a tyr residue in the B10 and a glut in the E7 positions, both of which are believed to participate in H-bonding to bound O2 that results in extremely slow off-rates. On the other hand, Chlamydomonas Hb is unusual in that the ferrous protein has a high-spin to low spin (six-coordinate) transition pK of 8.5, whereas the ferric protein has a high-spin to low spin (six-coordinate) transition pK of 6.4. This suggests that the distal ligand of the low spin forms of the ferrous and the ferric proteins may be different. The ferric protein exhibits a single low spin form with g values of 2.52, 2.31, 1.86, and thus an anisotropy similar to that of myoglobin hydroxide. However, 17O EPR and 18O resonance Raman studies failed to provide direct evidence for a hydroxide ligand. Spectroscopic studies on the distal mutants of the B10 Tyr, E7 Gln, and the E10 Lys residues have so far failed to identify a possible amino acid ligand. Further studies are being conducted to identify the distal ligand and to understand the unusual low spin EPR spectrum.